Introduction exercise (10). As exercise duration and intensity increase, the

adenine nucleotide (AdN) pool and the ATP/adenosine di- Skeletal muscles are the main organs used during exercise. Un- phosphate ratio decreases (7), which causes specific patterns and fortunately, performing muscle biopsies on highly trained athletes is unpractical, and other methods such as blood measurement magnitude of accumulation of these biomarkers in blood. must be used to reflect muscle biomarker concentration levels. In Biomarkers such as LA (3,5,8,9) and NH3 (1,10,11,18,19,31–34) have been extensively researched in difference stages of exercise addition, highly trained athletes also possess different levels of muscle mass which can affect magnitude of biomarker release. (1,11,15,18,19,32–34). New purine metabolite biomarkers have Because muscles use adenosine triphosphate (ATP) for energy been studied during and after exercise (13,22,30), after short-term production, measuring ATP metabolism biomarkers can help to training lasting 1–7 weeks (27–29), during a 1-year training cycle in better understand energy system utilization during different types highly trained athletes (36,38–40), and even considered a universal of training and exercise (1,3,10,11,20,33). Mechanisms related to indicator of training status (37) as well as a predictor of performance depletion and replenishing of ATP stores due to increased con- in highly trained athletes (35). To our knowledge, there is an in- sumption or decreased provision are reflected by blood concen- adequate amount of research, studying ATP metabolism biomarker tration of 3 basic biomarkers, i.e., lactate (LA), ammonia (NH3), efflux before, during exercise, and during the postexercise recovery and oxypurines (7), as well as decreased power output during period in highly trained athletes. Sjodin and Hellsten-Westing (24) studied only hypoxanthine (Hx) and uric acid (UA) before, during, Address correspondence to Jacek Zieliński, jacekzielinski@wp.pl. and after exercise only in 4 well-trained runners. Ogino et al. (19) Supplemental digital content is available for this article. Direct URL citations appear studied LA, NH3, and Hx before, during, and after exercise but only in the printed text and are provided in the HTML and PDF versions of this article on in healthy male volunteers. Finally, Stathis et al. (28) studied LA and the journal’s Web site (http://journals.lww.com/nsca-jscr). purine derivatives in 7 active but nonspecifically trained males with Journal of Strength and Conditioning Research 00(00)/1–9 the main focus on biomarker release during recovery as opposed to ª 2019 National Strength and Conditioning Association release during exercise. As shown, there is a lack of research

reflecting a greater range of ATP biomarker release around exercise of the transition period of their annual training cycle (beginning (rest, exercise, and recovery) in highly trained athletes in the same of preparatory phase). Controls, however, were not specifically training phase. Moreover, the contribution of specific muscle fibers, trained, recreationally performed common forms of physical ac- genetically predetermined or resulting from training adaptation, is of tivity (e.g., jogging, swimming, and team games) in their spare importance. Fast-twitch muscle fibers contain more ATP and phos- time, and did not exceed the 150 minutes of moderate-intensity phocreatine (16), are more glycolytically active (5) suited for LA physical activity per week recommended by the World Health production (8), and contain more AMP deaminase leading to greater Organization. The project was approved by the Ethics Committee AMP deamination producing more NH3 (13,33) and ultimately at the Poznan University of Medical Sciences and was performed more Hx in muscle compared with slow-twitch muscle fibers. Fur- according to the ethical standards laid down in the Declaration of thermore, in the context of exercise, there were no attempts to elu- Helsinki. Each subject was informed of the testing procedure, cidate the relationships between skeletal muscle mass (SMM) and purpose, and risks of the study and submitted their written con- ATP metabolism biomarker concentration. It is unknown whether sent to participate. changes in ATP metabolism biomarker concentrations are related to the changes in muscle mass. Greater muscle mass is related to the total amount of ATP-PCr in muscle that can be used during exercise Procedures and increases the amount of ATP in muscle that can be produced through anaerobic glycolysis (21). Because muscle mass levels and Subjects were instructed not to participate in any high-intensity or muscle properties are different in athletes of diverse physiological long-duration training sessions at least 24–48 hours before test- and training profiles, this could cause different release dynamics ing. Testing was performed in the morning hours, 3 hours after throughout exercise. a light breakfast (no coffee or tea). Before the exercise test, sub- The aim of this study was to determine changes in blood con- jects underwent body composition analysis. Afterward, an in- centration of ATP metabolism biomarkers (LA, NH3, and purine cremental treadmill exercise test until volitional exhaustion was metabolites) during graded exercise and in the recovery period, in performed. During all examinations, room temperature remained athletes of different training profiles and differences in SMM. We at 20–21° C. hypothesize that (a) high-level specialized sport training causes different adaptations resulting in specific exercise-related release Anthropometric and Body Composition Analysis. The anthro- dynamics of ATP metabolism biomarkers during standard exer- pometric measurements taken were body mass (kg) and height cise and in the postexercise recovery period, and that (b) differ- (cm) using a digital stadiometer (SECA 285; SECA, Hamburg, ences in muscle mass of athletes will affect biomarker release. Germany). Body mass index was calculated by dividing body mass by height squared. The dual X-ray absorptiometry method, using Lunar Prodigy Pro (GE Healthcare, Madison, WI, USA) Methods and enCORE v. 16 SP1 software, was used for body composition Experimental Approach to the Problem analysis. During examination, subjects only wore their under- garments, without jewelry and metal objects to minimize mea- To obtain the full-blood ATP biomarker spectrum of release surement error. Skeletal muscle mass was calculated based on around exercise, the testing procedures included an incremental regression models appropriate for age and sex (17). exercise test, where blood samples were obtained at rest, whereas subjects ran on a mechanical treadmill with increasing speed, and Respiratory Parameters. An incremental exercise test until voli- up to 30 minutes after exercise. Elite athletes of different sport tional exhaustion on a mechanical treadmill (H/P Cosmos Pulsar, specializations were chosen to represent diverse metabolic and Sports & Medical, Nussdorf-Traunstein, Germany) was per- physiological adaptations (speed-power, endurance, and mixed) formed to determine the maximal oxygen uptake (V̇ O2max). to see whether long-term specialized training would produce Initial speed was set at 4 km·h21 and increased after 3 minutes to different biomarker responses. Subjects underwent body com- 8 km·h21. After that point, treadmill speed increased pro- position analysis to obtain SMM levels. A control group of rec- gressively by 2 km·h21 every 3 minutes until volitional exhaus- reationally trained athletes was used to represent a group of tion. Once a 10-km·h21 speed was reached, blood samples were general training adaptations to exercise contrary to the highly drawn from the participant at the end of each 3-minute stage. specialized athletic groups. Respiratory parameters were measured (breath by breath) by an ergospirometer (Cortex Metamax 3B R2, Leipzig, Germany) and analyzed using MetasoftStudio v. 5.1.0 Software (Cortex- Subjects Metamax 3B R2; Cortex Biophysik, Leipzig, Germany). Heart Four groups of athletes participated in the study: sprinters (SP, rate (HR, b·min21) was monitored with a Polar Bluetooth Smart n 5 11) specialized in the 100 and 200 m events, aged 24.2 6 3.2 H6 (Polar Electro Oy, Kempele, Finland) HR monitor. All sub- (range 21–30) years, and practicing sport for 8.5 6 2.5 years, jects were familiar with the exercise protocol since they already endurance athletes (EN, n 5 16) consisting of triathletes and long- performed this test previously. distance runners aged 23.4 6 3.6 (range 18–31) years, and practicing sport for 8.7 6 1.9 years, futsal players (FU, n 5 12) Blood Sampling. Subjects wore a catheter (BD Venflon Pro 1.3 aged 24.5 6 3.8 (range 18–29) years, and practicing sport for 3 32 mm; Becton Dickinson, Helsingborg, Sweden) patent 10.0 6 3.4 years, and amateur runners (AM, n 5 12) representing with isotonic saline (0.9% NaCl), from which blood was drawn the control group aged 27.7 6 4.1 (range 22–33) years with no from one of the antecubital veins. Blood samples were collected past or current competitive sport history (recreational sports ac- at rest, at the end of each 3-minute stage above 10 km·h21 tivities 3–5 times per week). A more detailed description of the treadmill speed, immediately after exercise, and 5, 10, 15, subjects is presented in Table 1. Sprinters, EN, and FU were 20, and 30 minutes into the postexercise recovery period. members of the Polish National Team and were tested at the end Later, a 2.7-ml blood sample was taken into 2 monovettes

(S-Monovette 2.7 ml KE; Sarstedt, Nümbrecht, Germany) one Purines in plasma were determined by high liquid performance with a lithium anticoagulant (heparin) and another containing chromatography (HPLC) with UV detection as described earlier an anticoagulant (EDTA). (26). The analyses were performed using 1100 HPLC system (HPLC System; Agilent, Santa Clara, California). Separation was Lactate and Ammonia. Biosen C-line (EKF diagnostic GmbH, achieved with analytical column BDS Hypersil C18 (150 3 4.6 mm Barleben, Germany) was used to measure lactate level using 3 3 mm; Thermo ScientificTM, Waltham, Massachusetts) main- whole blood. To determine lactate level, 20 ml of whole blood tained at 18° C, protected by SecurityGuard precolumn (Secur- was placed into a capillary. The apparatus measurement accu- ityGuard precolumn; Phenomenex, Torrance, California). The racy (CV) was 1.5% for 12 mmol·L21 concentration. To de- mobile phase consisted of A: 122 mM KH2PO4, 150 mM KCL, termine ammonia level, PocketChem BA PA-4140 (Arkay, and 28 mM K2HPO4 and B: 15% (vol/vol) acetonitrile in A. The Kyoto, Japan) was used. To perform a measurement on a testing percentage of B changed from 0 to 100% in 8 minutes, was strip (Ammonia Test Kit II; Arkay), 20 ml of blood was placed maintained at 100% for 1 minute, and then returned to 0% for re- using a pipette. The apparatus measuring range is 8–285 equilibration. The separation time with re-equilibration was 13.5 mmol·L21. The apparatus measurement accuracy (CV) minutes and was conducted at a flow rate of 0.9 ml·min21. The was 2.3%. sample injection volume was 20 ml. The quantitative analyses were performed based on external calibration of the signal at 254 or 280 Purine Metabolites (Hx, X, UA). The vial (Eppendorf, nm. Data acquisition and processing was managed by the Chem- Wesseling-Berzdorf, Germany) containing the blood mixture station software (Chemstation Software; Agilent, Santa Clara, with EDTA was centrifuged (Universal 320R; Hettich Lab California). The within-run/between-run %CVs were 3.1/4.1, 3.3/ Technology, Tuttlingen, Germany) at 4° C for 30 seconds at 4.4, and 2.7/3.2% for Hx, X, and UA, respectively. 14,000 rpm (using short spin function). After centrifugation, 0.2 ml of plasma was pipetted into 1.5 ml of vials (Eppendorf, Statistical Analyses Wesseling-Berzdorf), in duplicate and immediately frozen in liquid nitrogen. All samples were then stored in a freezer (280° The sample size was a priori estimated based on the assumption C). At the time of extraction, 0.2 ml of 1.2 mol·L21 HClO4 was that effect size will be at least medium. Using an a-level of 0.05, added to frozen plasma for deproteinization. After centrifuga- a power (1 2 b) of 0.80, it was calculated that at least 8 par- tion, to remove the protein pellet an acid supernatant was ticipants would be needed to detect a significant change or dif- neutralized with 3 mol·L21 K3PO4, centrifuged to remove pre- ferences in lactate and plasma purine metabolite concentration cipitated KClO4, and stored in 280° C. (G*Power; Heinrich-Heine-Universitat Dusseldorf, Dusseldorf,

Germany). A one-way repeated-measures analysis of variance and X concentration was shifted by 5–10 minutes (even 15 (ANOVA) was performed to test the changes in LA, NH3, and minutes in controls) into recovery period (after exhaustion). In purine metabolite (Hx, X, and UA) concentration over time case of UA, its concentration in all groups still increased after 30 (exercise and recovery measurement points) within each group minutes of recovery, and thus actual maximal values could not of participants. A one-way repeated-measures ANOVA was also be measured. used to test differences between groups at the same measurement times. A post hoc Scheffe test was conducted if a significant difference was found (p , 0.05). All effect sizes for ANOVA Correlations Between Biomarker Concentration and Skeletal were calculated using h (2) and defined as small (0.01), medium Muscle Mass (0.06), and large (0.14) while confidence intervals (CI 95%) There were no significant correlations observed between SMM were also calculated. Pearson correlation coefficients (r) and and biomarker concentration at maximal intensity and recovery effects sizes were used to describe the relationship between within groups. Pearson’s correlation coefficients (r) during exer- biomarker concentrations within each group at maximal exer- cise and recovery for LA were 20.02 to 0.28, 20.13 to 0.15, cise and recovery compared with total-body SMM and defined 0.10–0.22, and 20.13 to 0.17 (FU, EN, SP, and AM, re- as small (0.1), medium (0.3), or large (0.5). All statistical anal- spectively), for NH3 were 20.2 to 0.27, 0.15–0.46, 0.16–0.30, yses were performed using STATISTICA 13.0 software (Stat- and 20.05 to 0.44 (FU, EN, SP, and AM, respectively), for Hx Soft, Tulsa, OK, USA). Significance level for all statistical were 20.13 to 0.18, 20.47 to 20.44, 20.45 to 20.03, and 0.25 analysis was set at p # 0.05. All values were presented as mean to 0.46 (FU, EN, SP, and AM, respectively), for X were 20.12 to 6 SD. 0.18, 20.47 to 20.42, 20.45 to 20.03, and 0.21 to 0.37 (FU, EN, SP, and AM, respectively), and for UA were 20.43 to 20.27, 20.11 to 0.19, 0.16–0.40, and 0.06–0.36 (FU, EN, SP, and AM, Results respectively). Descriptive Characteristics Sprinters had a significantly greater amount of SMM compared with the remaining groups and significantly greater weight at ex- Discussion amination than EN. EN differed significantly from the SP and AM This is the first study to measure ATP metabolism biomarker (LA, groups in maximal absolute (L·min21) and relative (ml·kg21·min21 NH3, and purine) concentration in blood at rest, during in- and ml·kg smm21·min21) V̇ O2max. The AM group was signifi- cremental exercise, and in the postexercise recovery period with cantly older than the other groups. Other baseline anthropometric many sampling points in highly trained athletes. Each group of and physical characteristics were similar in all groups and are our elite athletes represented a distinct physiological and meta- presented in Table 1. bolic training profile dictated by sport discipline. Sprinters rep- resented speed/power athletes engaged in very high-intensity exercise requiring mostly anaerobically dominant energy contri- Biomarker Concentration Changes bution (6). Triathletes and distance runners represented endur- All detailed mean values with SD, effects sizes, and confidence ance athletes performing predominantly aerobic exercise in their intervals are presented as supplementary digital content (see training (4). Futsal players represented the team sport group Tables 1 and 2, Supplemental Digital Content 1 and 2, http:// characterized by mixed energy system contribution from both links.lww.com/JSCR/A128 and http://links.lww.com/JSCR/ aerobic and anaerobic pathways (2). A129). Absolute LA concentration at rest, at the point of ex- Our main finding is that the pattern and magnitude of bio- haustion, and during recovery did not differ between groups. marker concentration depends on the specific training profile of During consecutive stages of exercise, sprinters showed highly trained athletes in distinct sport disciplines. At rest, during a rapid increase in LA from the initial stage of the test, whereas incremental exercise, and up to 30 minutes into the postexercise in endurance runners, the increase was noticeably delayed recovery period sprinters had lowest purine metabolism bio- (Figure 1A). After converting LA per 1 kg muscle mass, the marker concentrations, and endurance athletes had lowest am- groups also significantly differed at exhaustion and during monia concentrations. For LA during exercise, the lowest recovery. Highest LASMM values were obtained by endurance concentrations were noted in endurance athletes, except when athletes and lowest by sprinters (Figure 1B). reaching maximum intensity, where the differences between For absolute NH3 values, significant between-group differ- groups were not significant. In this study, sprinters had signifi- ences were observed at rest, during exercise, and recovery but not cantly greater absolute SMM than all remaining groups. Finally, at maximum exertion. During exercise, sprinters had a higher there were no correlations between SMM and biomarker con- concentration and most rapid increase in NH3 and endurance centrations at maximal intensity and during recovery within each athletes the lowest values compared with other groups, whereas group. during recovery, controls showed highest and endurance athletes We observed that groups differed in regard to specific bio- the lowest NH3 levels (Figure 1C). Converting the values relative marker concentration. Sprinters had overall lower purine bio- to SMM resulted in lowest values in sprinters during recovery marker concentration than all groups. Sprint training is mainly (Figure 1D). composed of short, high-intensity exercise bouts with complete or Across the entire measurement spectrum (rest, exercise, ex- incomplete rest depending on training goal (6,36). This type of haustion, and recovery), the groups significantly differed in both exercise causes a decrease in the muscle adenine nucleotide pool absolute and relative (per 1 kg muscle mass) values of Hx, X, (including ATP), through degradation of inosine monophosphate and UA. Regardless of metabolite and measure unit, highest (IMP) into Hx and X, which further effluxes into the bloodstream values were consistently obtained by controls and lowest values and forms UA, later being excreted with urine (13,14). Zieliński by sprinters (Figure 2A‒F). Unlike LA and NH3, maximum Hx et al. (36–40) have demonstrated that short-term and long-term

training decreases postexercise Hx levels, which coincides with change differences between groups compared with absolute val- increased activity of the enzyme hypoxanthine-guanine phos- ues. This demonstrates that blood purine metabolite concentra- phoribosyltransferase (HGPRT) that catalyzes the conversion of tion is not sensitive to differences in SMM but only to specific Hx to IMP. This Hx decrease occurs during periods of training metabolic adaptations. with high-intensity intermittent-type sessions, especially during In terms of NH3, endurance athletes overall had lower blood the special preparatory and competitive period. Also, this is more concentrations than all groups. Endurance training is mostly evident in highly trained athletes, since they engage in more composed of high-volume, low- to moderate-intensity-type ex- metabolically challenging training sessions producing greater ercise bouts with some higher-intensity training sessions in- ATP precursor loss (36,37,39). Thus, high-intensity intermittent corporated into certain phases of the annual training plan training results in higher erythrocyte HGPRT activity (36–40) (4,36,38). Endurance training has been shown to reduce ammo- causing more efficient utilization of the salvage pathway in re- nia production during submaximal exercise suggesting that en- covering derivatives of adenine nucleotide breakdown, limiting durance training does not decrease ammonia production capacity purine efflux from skeletal muscle, and, consequently, decreasing but instead delays it (33). Endurance training also increases the postexercise plasma Hx concentrations (12,14,28,36–40). Futsal mitochondrial content and blood flow capacity of muscles players also had relatively lower purine concentrations at rest, resulting in less adenosine monophosphate (AMP) deaminase during exercise, and after exercise compared with endurance activity and decreased ammonia production. This most likely athletes and controls. Since gameplay during matches requires the causes greater utilization of the aerobic system during this type of ability to repeat sprints with incomplete rest while continuously incremental exercise, thus decreasing overall ATP breakdown running, team sport training also encompasses higher-intensity through AMP degradation that is indicative of cellular energetic intermittent training interspersed with aerobic training sessions stress and cellular energy status (7). Endurance athletes also have (25,27). Plasma purine concentration relative to SMM did not a smaller amount of fast-twitch (FT) muscle fibers, which contain

more AMP deaminase associated with greater NH3 accumulation containing more AMP deaminase, leading to greater NH3 accu- in blood (33). Oxidative capacity of muscle has shown to be mulation in blood (33). Blood NH3 concentration relative to sensitive to training, and at the same exercise intensities, aerobi- SMM (NH3SMM) at maximum intensity and recovery was lowest cally trained muscle will have a smaller increase in glycolytic rate, in sprinters compared with absolute values. This is most likely due suggesting that AMP deaminase is less active in FT fibers of aer- to greater absolute SMM in sprinters. obically trained muscles (33). Sprint-trained athletes, on the other For LA, during exercise, the lowest concentrations were noted hand, possess higher amounts of fast-twitch (FT) muscle fibers, in endurance athletes, except when reaching maximum intensity,

where endurance athletes had the highest concentrations. Blood mass will seem to have lower biomarker levels at the same exercise lactate response to incremental and constant intensity tests dif- intensity, which could falsely imply better training level. ferentiates 3 intensity zones, which reflect 3 distinct metabolic Determining LA and NH3 plasma concentration is more fa- responses (3): (a) exercise intensity that does not require an in- vorable during exercise because both increase proportionately to crease in glycolytic ATP resynthesis (intensity below “lactate exercise intensity and achieve peak concentration at maximum threshold”), (b) exercise intensity that causes an increase in gly- intensity. Determining purine metabolite (Hx, X, and UA) con- colytic rate leading to blood lactate concentration steady state, centration is more useful after exercise because purine efflux is matched by pyruvate utilization and therefore leading to intensity indicative of adenine nucleotide loss and consequently reflects en- between lactate threshold and maximal lactate steady state ergy loss. Determining LA threshold is often used in endurance (MLSS), and (c) exercise intensity leading to a glycolytic rate that athletes to prescribe appropriate training loads below, at, or above cannot be matched by aerobic pyruvate utilization indicating this threshold (23). However, because endurance athletes have LA need for ATP generation through anaerobic glycolysis (intensity thresholds at relatively high intensities (percentage of their above MLSS). Training of highly trained endurance athletes is V̇ O2max), and because most engage in low- to moderate-intensity mostly dedicated to the first intensity domain (70–90% of exercise below this threshold, it leaves a large unquantifiable area training volume) and less to the second and third intensity to prescribe training intensity. Therefore, when quantifying train- domains (1). This most likely leads to LA concentrations being ing load at intensities below LA threshold, NH3 is more intensity- very low during initial stages of an incremental exercise test. Also, sensitive. LA is a more adequate marker to assess exercise intensity endurance athletes’ LA thresholds are relatively high, which in sprint-trained and game sport athletes, and mainly during allows them to exercise at a higher relative intensity (%V̇ O2max) higher-intensity interval–type sessions (HIIT), since it is an in- for longer duration. In endurance athletes, glycolytic rate is dicator of anaerobic glycolysis utilization during exercise (7). matched by pyruvate utilization at much higher intensities than in NH3 may be used as an indirect indicator of muscle ATP loss untrained and recreationally trained subjects (3). This may in- during exercise (11) because it increases proportionately to ex- dicate that lactate shuttling to other tissues that use lactate as fuel ercise intensity and is more intensity-sensitive at lower intensities in the body may be higher in these athletes, which is reflected by below lactate threshold. This verifies the study by Banister et al. decreased LA blood concentration. Endurance athletes also have (1), where NH3 blood concentration was elevated even at work more slow-twitch (ST) muscle fibers, which produce less LA rates at 40–50% V̇ O2max, whereas LA blood concentration in- through anaerobic glycolysis. Sprinters have lower LA thresholds, creased much more slowly during exercise. Itoh (15) observed a greater percentage of fast-twitch (FT) muscle fibers therefore use that peak ammonia concentration did not differ between 15-, 30-, anaerobic glycolysis to a higher degree leading to greater LA and 45-second sprint bouts, but LA concentration did. This production and release. In addition, sprinters have greater levels demonstrates that ammonia is more intensity-sensitive because of PCr that can be used during exercise. Utilization of PCr in turn each sprint was performed at maximal volitional exertion leading is coupled closely with glycolysis; depending on intensity and to similar ammonia concentrations (depletion of TAN pool after duration of exercise, greater depletion of PCr will lead to in- 15 seconds in all cases). LA concentrations increased because of creased glycolysis (21). Finally, futsal athletes had the second increases in utilization of anaerobic glycolytic pathways in longer largest LA concentration during incremental exercise (sprinters sprint bouts. Determining plasma NH3 concentration can be used the most). Since futsal athletes represent mixed energy system to evaluate, monitor, and prescribe exercise intensity during contribution, their LA concentration increase could be caused by clinical exercise tests, conditioning programs, occupational tasks, greater anaerobic glycolytic activity than endurance athletes and and athletic performance. controls, which relied more on aerobic contribution. For LAmax, Purine metabolites, especially Hx can be used during exercise sprinters had the lowest concentrations relative to SMM because Hx increases proportionately to exercise intensity, but it (LASMM). In contrast to absolute LA concentration, LASMM is worthwhile to measure Hx concentration in the postexercise concentration during exercise was lowest in sprinters compared recovery period to determine total purine derivative loss. This can with other groups most likely due to greater absolute SMM. help assess recovery needs of athletes after high-intensity exercise LA, NH3, and Hx are all valuable for monitoring training sessions. Hx level also indicates adaptation to high-intensity ex- status in highly trained athletes, and each demonstrates different ercise because of reduced purine efflux from skeletal muscle and exercise-induced changes in skeletal muscle. Gorostiaga et al. (11) can be viewed as a marker of anaerobic metabolism (14,36). Hx found that LA concentration in muscle correlated with plasma LA can be used in highly trained athletes as an indicator of training concentration in blood, and that ATP concentration in muscle status in different training phases of a 1-year training cycle re- correlated with plasma NH3 concentration in blood. This indi- gardless of age and sport discipline (37). Using Hx is particularly cates that by measuring plasma concentration of LA or NH3, valuable in highly trained athletes because commonly used energy status of skeletal muscle can be indirectly determined. measures such as V̇ O2max, “anaerobic threshold” or LA con- Purine concentration, on the other hand, mirrors adenine nucle- centration are not sensitive to changes in training status and otide derivative loss in muscle. Stathis et al. (29) and Sahlin et al. performance of highly trained athletes. Training prescription (22) demonstrated that subjects with a high blood and muscle should be revised based on recent knowledge regarding purine purine concentration exhibited a decreased muscle total adenine metabolism adaptations to fit training programs of highly trained nucleotide (TAN) pool or muscle ATP level. Overall, plasma LA athletes. Hx can also be used during tapering periods to determine concentration reflects muscle LA concentration that further preparedness and readiness to competition (37). Notably, UA reflects anaerobic glycolysis utilization, plasma NH3 concentra- plasma concentration did not reach peak values during exercise tion reflects muscle ATP content, and plasma purine concentra- and even after 30 minutes after exercise. This questions its use- tion reflects TAN pool depletion. However, calculating fulness as a biomarker of muscle status since it is difficult to biomarker concentration relative to muscle mass (per 1 kg of achieve a peak value in the specified time frame. SMM) does not present a clear and accurate picture and does not Finally, there were also no correlations observed between have diagnostic value. Athletes having larger amounts of muscle SMM and biomarker concentrations at maximal intensity and

recovery in all groups. This indicates that muscle mass levels are Acknowledgments not significantly related to magnitude of biomarker release in The authors do not have professional relations with any blood. Because we did not perform muscle biopsies, we were not companies or manufacturers who will benefit from the results able to determine biomarker concentrations in muscle and their of this study. The results of this study do not constitute correlations with blood biomarker levels. However, this may in- endorsement of the product by the authors or the NSCA. The dicate that training level and especially training profile as a result authors thank the coaches, athletes, and volunteers for their full of metabolic adaptations in muscle dictates pattern of biomarker participation in the study. The authors declare that they have no release in blood, not muscle mass itself. Although more muscle conflicts of interest. This work was funded by the Polish Ministry mass may indeed produce greater absolute amounts of these of Science and Higher Education from financial resources of the biomarkers in muscle, the blood concentration is the combined National Science Center within the OPUS 5 program (application result of many factors (production and utilization in various and grant number: 2013/09/B/NZ7/02556). metabolic processes). A limitation in this study was that subjects could not perform an additional familiarization session with V̇ O2max testing a few days before the actual exercise (with blood sampling). Because most athletes are part of the Polish National Team, it is difficult to ask References them to perform additional tests since they have a very rigorous 1. Banister EW, Allen ME, Mekjavic IB, Singh AK, Legge B, and Mutch training cycle, and additional loading in the form of this exercise BJC. 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